Animal protein intake is directly associated with serum level of pentraxin 3 in hemodialysis patients

Inflammation plays an important role in Cardiovascular disease (CVD) pathogenesis as the main cause of mortality in hemodialysis (HD) patients. Despite the relevance of nutrition and dietary intakes for inflammation status, the role of dietary protein sources remains unclear. The aim of this study was to evaluate the association between the different types of dietary protein and pentraxin 3 (PTX3) levels in HD patients. In this multi-center cross-sectional study, 227 adult patients undergoing HD for a minimum 90 days were recruited. A validated 168-item food frequency questionnaire was used to assess dietary intakes. Also, 5 ml blood samples were collected from each patient to measure the concentration of serum PTX3. Overall, 227 patients, including 63 women and 164 men, with a mean age of 58 years, participated in this study. There was a greater intake of animal protein per kilogram dry weight among patients with higher levels of PTX3 (0.46 vs. 0.54 g/kg; P = 0.035). In contrast, consumption of total protein and plant protein per kilogram dry weight was not different across PTX3 levels. Moreover, the chance of increased PTX3 concentration was directly associated with a one-unit increase in animal protein intake per kilogram dry weight, after adjusting for confounders. We did not observe any association between one-unit increases in plant protein intake per kilogram dry weight and chance of increased PTX3. In conclusion, animal protein intake was directly associated with circulating PTX3.

Nutrition and dietary intakes are among the most important factors influencing the inflammation status in HD patients 16 .Malnutrition, as a cause of inflammation, is prevalent among these patients, resultant from decreasing amounts of dietary protein intake to slow down progression of chronic kidney disease (CKD) 17 .Nevertheless, the role of dietary protein sources in inflammation status has been neglected 18 .Results of a recent meta-analysis demonstrated a favorable relationship between a healthy dietary pattern (i.e., consuming more plant proteins and fish, along with less red meat) and mortality risk in CKD patients 19 .Moreover, a vegetarian diet can be recommended to adults with CKD because it can provide more protein with less bioavailable phosphorus 20 , and reduce uremic toxin levels 21 , hypertension 22 , metabolic acidosis 23 , and inflammation 24 .A systematic review and meta-analysis reported that in comparison with total protein intake, animal proteins elevated CRP concentration.Also, a decreasing trend in CRP was reported when plant proteins compared with animal proteins in adults with CKD 25 .Although previous studies have showed that animal protein had adverse effects on CRP concentration, the association between the sources of dietary protein and levels of PTX3 has not been assessed in HD patients.Therefore, we aimed to evaluate the association between different types of dietary protein (animal vs. plant) and levels of circulating PTX3.

Sample size calculation
Sample size was estimated by following formula 26 : N = [(Z 1−α/2 ) 2 × SD 2 ]/day 2 .PTX3 was used as the main variable to calculate the sample size.According to previous studies conducted in Iran on HD patients, the standard deviation of PTX3 was equal to 2.5 ng/ml 27 .Also, α was defined as 0.05, and d = 0.33 ng/ml.Therefore, minimum required sample size for this study was 221.

Study design and population
From September 2021 to March 2022, we carried out a multi-center cross-sectional investigation on adult maintenance HD patients.Individuals were selected from five different HD centers in Isfahan, Iran.Those who met the following criteria were included: (1) being on maintenance HD for a minimum of 90 days; (2) aged 18 or older and any gender; and (3) being able and eager to take part in survey.In contrast, patients were excluded if they: (1) had enteral or parenteral nutrition; and (2) reported daily energy intakes lower than 800 kcal/day (3347 kJ) or more than 4200 kcal/day (17,572.8kJ) (because of over-under reporting) 28 ; (3) were smokers; and (4) recorded history of myocardial infarction.Informed consent was obtained from all participants, after a brief description of the study's significance and protocol, prior to study commencement.The study protocol was approved by The Research Ethics Committee of Isfahan University of Medical Sciences, Isfahan, Iran (IR.MUI.RESEARCH.REC.1399.605).All methods were performed in accordance with the relevant guidelines and regulations.

Dietary assessment
The usual dietary intake during the past year was evaluated using a 168-item semi-quantitative food frequency questionnaire (FFQ).FFQs were filled out via in-person interviews by an experienced dietitian.This questionnaire contained a standard portion size for each food item along with nine options for frequency of consumption on a daily, weekly or monthly basis.Finally, analyzing dietary intakes was performed by converting reported food intakes to gram per day using household measures 29 .Then measuring macro-and micro-nutrients intake was performed by Nutritionist IV software (First Databank, Hearst Corp).Previously, the validity and reliability of this questionnaire were assessed and shown to be acceptable in the Iranian population 30 .

Laboratory assessments
A venous blood sample (5 mL) was collected from each participant.Then, serum was separated by centrifuging (2000 rpm for 10 min at 4 °C).The concentration of serum PTX3 was measured by using enzyme-linked immunosorbent assay (ELISA) kits (ZellBio GmbH, Germany) based on the Biotin double antibody sandwich technology (Inter Assay PTX3: CV < 10%, Intra Assay PTX3: CV < 12%).

Anthropometric measurements
To measure dry weight and height, we used a Seca scale and inelastic tape, respectively (Seca Co., Hamburg, Germany).Weight measurements were performed when patients had light clothes, measured to the nearest 0.1 kg.Also, height was assessed when subjects were in standard, upright position, and unshod, measured to the nearest 0.1 cm (cm).Dry weight was defined as the minimum acceptable weight following the dialysis session without exhibiting any signs or symptoms of hypovolemia or hypervolemia 31 .In order to calculate the body mass index (BMI), dry weight was divided by squared height.BMI < 23 kg/m 2 was the criterion of the malnutrition 32 .Addition, mid-upper arm circumference (MUAC), waist circumference (WC), as well as hip circumference (HC), were measured by an inelastic tape, with 0.1 cm accuracy, with participants in a standing position.The MUAC measurement was performed at an equidistant point between the inferior border of the acromion process and the tip of the olecranon process on the bare left arm 33 .For WC, the tape was placed at the midpoint between the iliac crest and lowest rib 34 and HC was obtained at the maximum circumference of the buttocks 35 .The waist-tohip ratio (WHR) was calculated by dividing the WC by the HC.

Assessment of Quality of HD:
To assess dialysis adequacy, Kt/V and Urea reduction ratio (URR) values were used.URR was calculated using the following equation 36 :

Assessment of other variables
Patients' general characteristics, such as age, job, and marital status, were obtained through verbatim questions.
In addition, the medical information, including causes of renal disease, comorbidities, dialysis duration, the number of dialysis sessions per week, dialysis vintage, as well as medications, were collected from hospital records.

Statistical analysis
The normal distribution of data was evaluated by Q-Q plot, histogram chart, Skewness statistic, and Kolmogorov-Smirnov test.Numerical variables were presented as mean ± standard deviation (SD), whereas categorical data were shown as numbers (percentages).Analyzing categorical and continuous variables across PTX3 levels was carried out using Chi-square tests and one-way analysis of variance (ANOVA), respectively.The association between PTX3 and intake of different types of dietary protein in HD patients was assessed using logistic regression.A wide range of cutoffs for PTX3 are reported for preventing different diseases.For example, in sever sepsis and fatal disease in bacteremic PTX3 cutoffs are 14.1 ng/ml and 15 ng/ml, respectively 37,38 .In HD patients, the best reported cutoff for PTX3 to predict morbidity and mortality were 0.55 ng/ml and 0.25 ng/ml, respectively 39 .In our study, the lowest concentration of PTX3 was 1.15 ng/ml.Therefore, we could not use this cutoff.As there is not an approved cut-off point for PTX3 in HD patients, we use the median-cut method.The odds ratios (ORs), with 95% confidence interval (CI), was reported for different adjusted models.In the first model, general confounders including age and sex were adjusted.As the independent variable (dietary protein intake) was strongly depends on energy intake, we also included calorie intake as a covariate in Model 1.In Model 2, we included remained variables based on the literature review including dialysis frequency, dialysis duration, urea reduction ratio, waist circumference, hip circumference, arm circumference, height, and cause of renal disease.We used SPSS version 21 for all statistical analyses.P < 0.05 was considered to be statistically significant.

Ethics approval and consent to participate
This study was ethically approved by The Research Council and Ethical Committee of Isfahan University of Medical Sciences, Isfahan, Iran, (Code: IR.MUI.RESEARCH.REC.1399.605).Also, all participants completed an informed consent form.Mohammed Hossein Rouhani as the lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.
Mean intake of different types of dietary protein per kilogram dry weight across PTX3 levels is presented in Table 3. Patients with higher levels of PTX3 consumed significantly more animal protein per kilogram dry weight (0.46 vs. 0.54 g/kg; P = 0.035).Intake of total protein and plant protein per kilogram dry weight was not different across levels of PTX3.
Results of the association between chance of elevated PTX3 and one-unit increase in intake of different types of dietary protein per kilogram dry weight are reported in Table 4.Although a one-unit increase in intake of total protein intake per kilogram dry weight was not associated with the chance of increased PTX3 in crude model and Model 1, it was marginally, significantly related to the chance of elevated PTX3 levels in Model 2 (OR = 3.114; 95% CI 0.943, 10.283; P = 0.062).Also, we observed a direct association between a one-unit increase in intake of animal protein intake per kilogram dry weight and chance of increased PTX3 concentration in the Model 1 (OR = 3.123; 95% CI 1.077, 9.053; P = 0 0.036) and Model 2 (OR = 4.524; 95% CI 1.247, 16.417; P = 0.022).A one-unit increase in intake of plant protein intake per kilogram dry weight were not related to the chance of increased PTX3, before or after adjusting for potential confounders.

Discussion
In accord with the aim of this study, we found that subjects with higher levels of PTX3 consumed significantly more animal protein per kilogram dry weight.Also, a one-unit increase in intake of animal protein per kilogram dry weight was directly associated with chance of increased PTX3.www.nature.com/scientificreports/Permanent low-grade inflammation is a concern in ESRD 40 , which can lead to exacerbated mortality and morbidity risk 41 .The inflammation experienced by CKD patients is traditionally monitored via interleukin-6, tumor necrosis factor alpha, and CRP [42][43][44] .Compared to these inflammatory biomarkers, PTX3 is considered to be a better predictor of inflammation due to its expression in wide range of tissues 45 .Also, PTX3 concentration is positively related to conventional inflammatory biomarkers 45 .In contrast to CRP, release of PTX3 occurs quickly from neutrophil granules in response to inflammatory signals 46 .The sensitivity of PTX3 to a micro inflammatory process is greater than other inflammatory biomarkers in HD patients 47 .Thus, PTX3 is accepted as a quick and sensitive indicator of dialysis-related inflammation among HD patients 47 .
Despite the importance of dietary protein intake in HD patients, the Kidney Disease Outcomes Quality Initiative (KDOQI) did not declare any recommendation regarding the type of protein (plant versus animal) due to the insufficiently powered studies 48 .Nephrologists typically avoid vegetarian-based diets 49 , as it has been traditionally believed that these kinds of diets are not only nutritionally inadequate, but also dangerous due to their high potassium contents 50 .Nevertheless, there is strong evidence advocating that plant based diets can be nutritionally sufficient and beneficial if they are well-balanced and varied [51][52][53] .On the other hand, no studies have demonstrated significant differences in serum potassium levels in a comparison of potassium intakes from plant based diets versus omnivorous diets 50 .Furthermore, some studies have shown the beneficial effects of plant protein or plant-based diets, including Dietary Approaches to Stop Hypertension (DASH), Mediterranean, or vegetarian diets, in reduction of inflammatory markers, as well as improvement of complications related to various chronic diseases 54,55 .Plant-based diets are rich in antioxidants and vitamins, whereas animal protein-rich Table 1.General characteristics of hemodialysis patients across levels of pentraxin 3. Data are presented as mean ± SD for continuous and percent for categorical variables.P-value obtained from chi-square analysis for categorical variables and Independent t-test for continuous variables.URR Urea reduction ratio. 1  www.nature.com/scientificreports/diets, particularly red and processed meat, generally contain high levels of sodium and saturated fatty acids, which can negatively impact on kidney functions 56 .Results of a recent meta-analysis on CKD patients revealed a positive association between consumption of animal protein and CRP concentration 25 .In contrast, results regarding other biomarkers of inflammation are controversial.Indeed, a previous investigation reported that animal protein may reduce the concentration of pro-inflammatory adipokines including chemerin and progranulin 57 .Therefore, it is necessary to perform a comprehensive study regarding the association between type of dietary protein and biomarkers of inflammation to elucidate a pattern that shows an overall conclusion.It would also be prudent to determine which inflammatory biomarkers are the most reliable predictor(s) of adverse outcomes in HD patients.There are several posited mechanisms regarding the association between inflammation and animal/plant protein intake.First, in comparison to plant protein, animal protein intake can negatively impact on the gut microbiome composition by producing greater amount of ammonia and sulfur-based materials.As a result of gut microbiome imbalance, promotion of inflammation will occur [58][59][60][61] .While a plant-based diet increases the production of short-chain fatty acids, which have beneficial effects on improvement of the dysbiotic microflora compositions, they can result in the inhibition of the pathogens growth, reduction of pro-inflammatory parameters, decreased oxidative stress, and lower uremic toxins 62,63 .Second, the cholesterol derived from animal fat and meat plays an important role in the development of inflammation [64][65][66] .PTX3 expression can induce both locally and systemically by circulating levels of LDL cholesterol (a pivotal mediator of atherosclerosis) 67 .Third, sulfur-containing amino acids found in animal proteins can enhance dietary acidity and result in exacerbating metabolic acidosis in CKD patients 68 .Dietary acid load can induce inflammation 69 .Meanwhile, metabolization of plant proteins is associated with a greater consumption of hydrogen ions 70 and greater production of bicarbonate in order to minimize acid productions 71 .Consequently, production of inflammatory markers, oxidative stress, and uremic toxins may be reduced through this process 72,73 .
We have presented a novel addition to the literature, highlighting that the type of protein consumed should be acutely considered in HD patients.Nevertheless, addressing the limitations associated with the present study is important.First, despite controlling for multiple confounders, residual confounders may remain, and can only be elucidated in further work.Second, the cross-sectional design of this study precludes causal inferences being made.Concomitant to the limitations, there are several strengths that should be noted.Indeed, the use of an appropriate biomarker, consideration of potential confounders, the relatively large sample size, and the multicenter design of the study are inherent strengths of the present study.Reporting the results on a per kilogram dry weight basis is another advantage of this study.As all HD guidelines recommend a certain amount of protein per kilogram, our findings can be comparable with guidelines.

Practical application
In conclusion, our findings suggest that, unlike consumption of plant protein, animal protein intake was significantly associated with increased circulating PTX3 levels among HD patients.Accordingly, extra consideration should be given the source(s) of protein intake in HD patients under clinical supervision.

Table 2 .
Dietary intake of hemodialysis patients across levels of pentraxin 3. Data are presented as mean ± SD.P-value obtained from analysis of covariance (ANCOVA) adjusted for total calorie intake.

Table 3 .
Mean intake of different types of dietary protein per kilogram dry weight across levels of pentraxin 3 in hemodialysis patients.Data are presented as mean ± SD.P-value obtained from analysis of covariance (ANOVA) adjusted for total calorie intake.

Table 4 .
The association between odds ratio for elevated pentraxin 3 (> Median) and one-unit increase in intake of different types of dietary protein per kilogram dry weight in hemodialysis patients.P-value obtained from logistic regression.Model 1 was adjusted for age, sex, total calorie intake.Model 2: was adjusted for age, sex, total calorie intake, dialysis frequency, dialysis duration, urea reduction ratio, waist Circumference, hip Circumference, arm Circumference, height, cause of renal disease.